The present invention relates to the field of detection and identification of Human Papillomavirus (HPV) infections in clinical samples.
Cervical cancer is the second most common malignancy in women, following breast cancer. Carcinoma of the cervix is unique in that it is the first major solid tumor in which HPV DNA is found in virtually all cases and in precursor lesions worldwide.
Nowadays, 74 HPV genotypes have been characterized and are numbered in chronological order of isolation. HPV is epitheliotropic and infects only the skin (cutaneous types) or the mucosa of the respiratory and anogenital tract (mucosal types). Thirty-six of the 74 HPV types are known to infect the uterine cervix. Based on the induced benign, premalignant or malignant lesions, HPV is divided into low-risk (e.g., HPV types 6, 11, 42, 43 and 44) and high-risk types (e.g., types 16, 18, 31, 33 and 45), respectively. The high-risk types account for more than 80% of all invasive cervical cancers. Consequently, detection and identification of HPV types is very important. The high-risk types are more consistently found in high grade SIL (Squamous Intraepithelial Lesion) and carcinoma in-situ than low-risk types which are mainly found in low grade SIL. This epidemiological observation is supported by molecular findings. For instance, the E6 and E7 proteins from low-risk types 6 and 11 bind p53 and pRB too weakly to immortalize keratinocytes in vitro or to induce malignant transformation in vivo (Woodworth et al., 1990). The circular ds-DNA genome of low-risk HPV types remains episomal whereas the genome of high-risk HPV types is able to integrate into the human genome.
Screening for malignant and premalignant disorders of the cervix is usually performed according to the Papanicoloau (PAP) system. The cervical smears are examined by light microscopy and the specimens containing morphologically abnormal cells are classified into PAP I to V, at a scale of increasing severity of the lesion. This cytomorphological method is an indirect method and measures the possible outcome of an HPV infection. Therefore, HPV DNA detection and typing is of importance in secondary screening in order to select patients for monitoring (follow-up) and treatment. This means that cervical smears classified as PAP II (atypical squamous metaplasia) or higher classes should be analyzed for low-risk and high-risk HPV types. Follow-up studies have shown that only high-risk HPV types are involved in the progression from cytologically normal cervix cells to high grade SIL (Remminck et al., 1995). These results indicate that the presence of high-risk HPV types is a prognostic marker for development and detection of cervical cancer.
Detection of HPV Infections
Diagnosis of HPV by culture is not possible. Also diagnosis by detection of HPV anti-bodies appears to be hampered by insufficient sensitivity and specificity. Direct methods to diagnose an HPV infection are mainly based on detection of the viral DNA genome by different formats of DNA/DNA hybridization with or without prior amplification of HPV DNA. The polymerase chain reaction (PCR) is a method that is highly efficient for amplification of minute amounts of target DNA. Nowadays, mainly three different primer pairs are used for universal amplification of HPV DNA. Two of these primer pairs, MY11/MY09 and GP5/GP6, are directed to conserved regions among diffent HPV types in the L1 region (Manos et al., 1989; Van den Brule et al., 1990). The other primer pair, CPI/CPIIg, is directed to conserved regions in the El region (Tieben et al., 1993).
Typing of HPV Isolates
There are several methods to identify the various HPV types.
1. HPV DNA can be typed by PCR primers that recognize only one specific type. This method is known as type-specific PCR. Such methods have been described for HPV types 6, 11, 16, 18, 31 and 33 (Claas et al., 1989; Cornelissen et al., 1989; Falcinelli et al., 1992; Van den Brule et al., 1990; Young et al., 1989). The primers are aimed at the E5, L1, E6, L1, E2 and E1 regions of the HPV genome for types 6, 11, 16, 18, 31 and 33, respectively (Baay et al., 1996). The synthesized amplimer sizes vary from 217 bp to 514 bp.
2. Another method is general amplification of a genomic part from all HPV types followed by hybridization with two cocktails of type-specific probes differentiating between the oncogenic and non-oncogenic groups, respectively. A similar typing method has been described without prior amplification of HPV DNA. In the Hybrid capture assay (Hybrid Capture Sharp Assay; Digene, Silver Springs, Md.), each sample is tested for a group of xe2x80x9chigh-riskxe2x80x9d HPV types (16, 18, 31, 33, 35, 45, 51, 52 and 56) and for another group of xe2x80x9clow-riskxe2x80x9d HPV types (6, 11, 42, 43 and 44) (Cox et al., 1995).
At present, classification of human papillomaviruses can be performed for instance by sequence analysis of a 450 bp PCR fragment synthesized by the primers MY11/MY09 in the L1 region (Chan et al., 1995) or by the primers CPI and CPIIg in the E1 region (Tieben et al., 1993). Phylogenetic analysis of these sequences allows classification of the different HPV types. By definition, if the sequence differences between two HPV isolates is higher than 10% they are classified as different types. Consequently, if the sequence differs more than 10% from any known HPV type it is classified as a novel HPV genotype. HPV isolates that differ between 2-10% are classified as different subtypes. Finally, if the sequence variation is below 2%, the 2 isolates are classified within the same subtype as different variants.
Aims of the Invention
It is an aim of the present invention to provide a rapid and reliable method for detection and/or identification of HPV, possibly present in a biological sample.
It is more particularly an aim of the present invention to provide a method for detection and/or identification of HPV comprising amplification of a polynucleic acid fragment of HPV and subsequent hybridization of this fragment to suitable probes.
It is also an aim of the present invention to provide a number of oligonucleotide primers and probes enabling said method of detection and/or amplification of HPV.
It is also an aim of the present invention to provide new HPV sequences.
It is furthermore an aim of the present invention to provide protocols according to which said amplification and hybridization steps can be performed. One format for the hybridization step is, for instance, the reverse hybridization format, and more particularly the LiPA technique.
It is also an aim of the present invention to compose diagnostic kits comprising said primers and probes, permitting the rapid and reliable detection and/or identification of HPV possibly present in a biological sample.
All the aims of the present invention are met by the following specific embodiments.
The present invention provides a method for detection and/or identification of HPV, possibly present in a biological sample, comprising the following steps:
(i) amplification of a polynucleic acid fragment of HPV by use of:
a 5xe2x80x2-primer specifically hybridizing to the A region or B region of the genome of at least one HPV type, said A region and B region being indicated in FIG. 1, and
a 3xe2x80x2-primer specifically hybridizing to the C region of the genome of at least one HPV type, said C region being indicated in FIG. 1;
(ii) hybridizing the amplified fragments from step (i) with at least one probe capable of specific hybridization with the D region of at least one HPV type, said D region being indicated in FIG. 1.
According to one preferred embodiment of the present invention, said probe mentioned in step (ii) is capable of specific hybridization with the D region of the genome of only one HPV type, and thus enables specific identification of this HPV type, when this type is present in a biological sample.
According to another preferred embodiment of the present invention, said probe mentioned in step (ii) is capable of specific hybridization with the D region of more than one HPV type, and thus enables detection of any of said more than one HPV type, when any of said types is present in a biological sample.
According to another preferred embodiment of the present invention, the 3xe2x80x2-end of said 5xe2x80x2-primer specifically hybridizing to the A region of the genome of at least one HPV type, is situated at position 6572 of the genome of HPV 16, or at the corresponding position of any other HPV genome, as indicated in FIG. 1.
According to another preferred embodiment of the present invention, the 3xe2x80x2-end of said 5xe2x80x2-primer specifically hybridizing to the B region of the genome of at least one HPV type, is situated at position 6601 of the genome of HPV 16, or at the corresponding position of any other HPV genome, as indicated in FIG. 1.
According to another preferred embodiment of the present invention, the 3xe2x80x2-end of said 3xe2x80x2-primer specifically hybridizing to the C region of the genome of at least one HPV type, is situated at position 6624 of the genome of HPV 16, or at the corresponding position of any other HPV genome, as indicated in FIG. 1.
According to another preferred embodiment of the present invention, said probe capable of specific hybridization with the D region of the genome of only one HPV type, more particularly specifically hybridizes to the E region, with said E region being a subregion of the D region, as indicated in FIG. 1.
According to another preferred embodiment of the present invention, said probe capable of specific hybridization with the D region of the genome of only one HPV type, more particularly specifically hybridizes to the 22 bp region situated between the B region and the C region, as indicated in FIG. 1.
According to another preferred embodiment, said 5xe2x80x2-primer specifically hybridizing to the A region of the genome of at least one HPV type, is chosen from the following list:
SGP3, SGP3A, SGP3B, SGP3C, SGP3D, SGP3E, SGP3F, SGP3G.
The sequences of said primers are shown in table 1 and in table 4.
According to another preferred embodiment, said 5xe2x80x2-primer specifically hybridizing to the B region of the genome of at least one HPV type, is chosen from the following list:
SGP1, SGP1A, SGP1B, SGP1C, SGP1D.
The sequences of said primers are shown in table 1, in table 4 and in table 11.
According to another preferred embodiment, said 3xe2x80x2-primer specifically hybridizing to the C region of the genome of at least one HPV type, is chosen from the following list:
SGP2, SGP2A, SGP2B, SGP2C, SGP2D, SGP2E, SGP2F, SGP2H, SGP2I, SGP2J, SGP2K, SGP2L, SGP2M, SGP2N, SGP2P.
The sequences of said primers are shown in table 1, in table 4 and in table 11.
According to another preferred embodiment, said probe capable of specific hybridization with the aforementioned 22bp region of only one HPV type, is chosen from the following list:
HPV6 Pr1, HPV6 Pr2, HPV6 Pr3, HPV6 Pr4, HPV6 Pr5, HPV11 Pr1, HPV11 Pr2, HPV11 Pr3, HPV11 Pr4, HPV11 Pr5, HPV16 Pr1, HPV16 Pr2, HPV16 Pr3, HPV16 Pr4, HPV16 Pr5, HPV18 Pr1, HPV18 Pr2, HPV18 Pr3, HPV18 Pr4, HPV18 Pr5, HPV31 Pr1, HPV31 Pr2, HPV31 Pr3, HPV31 Pr4, HPV31 Pr5, HPV31 Pr21, HPV31 Pr22, HPV31 Pr23, HPV31 Pr24, HPV31 Pr25, HPV31 Pr26, HPV31 Pr31, HPV31 Pr32, HPV33 Pr1, HPV33 Pr2, HPV33 Pr3, HPV33 Pr4, HPV33 Pr5, HPV33 Pr21, HPV33 Pr22, HPV33 Pr23, HPV33 Pr24, HPV33 Pr25, HPV33 Pr26, HPV40 Pr1, HPV45 Pr1 (=SGPP68), HPV45 Pr2, HPV45 Pr3, HPV45 Pr4, HPV45 Pr5, HPV45 Pr11, HPV45 Pr12, HPV45 Pr13, HPV52 Pr1, HPV52 Pr2, HPV52 Pr3, HPV52 Pr4, HPV52 Pr5, HPV52 Pr6, HPV56 Pr1, HPV56 Pr2, HPV56 Pr3, HPV56 Pr11, HPV56 Pr12, HPV58 Pr1, HPV58 Pr2, HPV58 Pr3, HPV58 Pr4, SGPP35, SGPP39, SGPP51 (=HPV51 Pr1), SGPP54, SGPP59, SGPP66, SGPP70 (=HPV70 Pr11), SGPP13, SGPP34, SGPP42, SGPP43, SGPP44, SGPP53, SGPP55, SGPP69, SGPP61, SGPP62, SGPP64, SGPP67, SGPP74 (=HPV74 Pr13), MM4 (=HPV4 Pr11), MM7, MM8, HPV18b Pr1, HPV18b Pr2, HPV31 Vs40-1, HPV31 Vs40-2, HPV31 Vs40-3, HPV34 Pr1, HPV35 Pr1, HPV35 Pr2, HPV35 Pr3, HPV39 Pr1, HPV42 Pr1, HPV42 Pr2, HPV43 Pr1, HPV43 Pr2, HPV43 Pr3, HPV44 Pr1, HPV44 Pr2, HPV44 Pr3, HPV44 Pr4, HPV45 Pr5, HPV51 Pr2, HPV53 Pr1, HPV54 Pr1, HPV54 Pr11, HPV54 Pr11as, HPV54 Pr12, 11HPV55 Pr1, HPV55 Pr11, HPV55 Pr12, HPV55 Pr13, HPV56 Vs74-1, HPV59 Pr1, HPV59 Pr11, HPV59 Pr12, HPV59 Pr13, HPV66 Pr1, HPV67 Pr1, HPV 67Pr11, HPV67 Pr12, HPV67 Pr13, HPV67 Pr21, HPV67 Pr22, HPV67 Pr23, HPV68 Pr1, HPV68 Pr2,HPV68 Pr3, HPV68 Vs45-1, HPV68 Vs45-2, HPV70 Pr1, HPV70 Pr12, HPV70 Pr13, HPV74 Pr1, HPV74 Pr11, HPV74 Pr12, HPV74 Pr2, HPV74 Pr3, HPVM4 Pr1, HPVM4 Pr12, HPVM4 Pr21, HPVM4 Pr22.
The sequences of said probes are shown in table 7 and table 12.
It is to be understood that combinations of the aforementioned embodiments are also preferred embodiments, for instance a method characterized in that said 5xe2x80x2-primer specifically hybridizing to the A region is chosen from the aforementioned respective list and that said 3xe2x80x2-primer specifically hybridizing to the C region is chosen from the aforementioned respective list.
It is an important feature of the present invention that the amplified polynucleic acid fragments of HPV fall within a short region of the L1 gene, a region that presents a high degree of sequence variability. Said region is denoted D region and for any HPV type consists of the region corresponding in a sequence alignment to the region from position 6553 to position 6646 of the genome of HPV 16, with the numbering being according to isolate PPH16, with Genbank accession number K02718. The advantage of amplifying a short fragment is that higher sensitivity can be obtained, i.e. a lower number of copies of HPV polynucleic acids can be detected and/or identified. The aforementioned primers may be used to amplify a fragment of approximately 65 bp (by use of 5xe2x80x2-primers specifically hybridizing to the B region and 3xe2x80x2-primers specifically hybridizing to the C region) or a fragment of approximately 94 bp (by use of 5xe2x80x2-primers specifically hybridizing to the A region and 3xe2x80x2-primers specifically hybridizing to the C region). However, it is obvious to one skilled in the art that other primers may be used in order to amplify other fragments within or overlapping with said D region. Preferred primers are shown in table 1 and in table 4. These primers permit amplification of polynucleic acid fragments of a large group of HPV types, but it may be desirable for some purposes to chose primers that selectively amplify a smaller group of HPV types.
The different types of HPV in a sample can be identified by hybridization of polynucleic acids of said types of HPV to at least one, preferably at least two, more preferably at least three, even more preferably at least four and most preferably at least five oligonucleotide probes. These probes may be designed to specifically hybridize to the D region of only one HPV genome, said D region being indicated in FIG. 1. Tables 7 and 12 contain a list of preferred probes specifically hybridizing to the 22 bp region within said D region, situated between the B region and the C region. These probes may be used together under the same conditions of hybridization and washing, for instance in a LiPA format (see below). Probes that have been optimized to work together in a LiPA format are. for instance the combination of HPV6 Pr1, HPV11 Pr1, HPV16 Pr1, HPV18 Pr1, HPV31 Pr25, HPV31 Pr31, HPV31 Pr32, HPV33 Pr21, HPV33 Pr25, HPV40 Pr1, HPV45 Pr11, HPV45 Pr12, HPV45 Pr13, HPV52 Pr5, HPV52 Pr6, HPV56 Pr11, HPV56 Pr12, HPV58 Pr2, HPV58 Pr3 and HPV58 Pr4 (see example 4), the combination of HPV6 Pr1, HPV11 Pr5, HPV16 Pr1, HPV18 Pr1, HPV18b Pr2, HPV31 Pr31, c31-3, HPV33 Pr21, HPV34 Pr1, HPV35 Pr1, HPV39 Pr1, HPV40 Pr1, HPV42 Pr1, HPV43 Pr3, HPV44 Pr1, HPV45 Pr11, HPV51 Pr2, HPV52 Pr5, HPV53 Pr1, HPV56 Pr12, c56-1, HPV58 Pr2, HPV59 Pr12, HPV66 Pr1, HPV68 Pr1, c68-1, HPV70 Pr12 and HPV74 Pr1, or the combination outlined in example 7. Probes specifically hybridizing to said 22 bp region should permit discrimination of all genital low-risk types including HPV types 6, 11, 34, 40, 42-44, 53, 54, 55, 59, 61, 62, 64, 67, 68, 71 and 74 as well as all genital high-risk types including HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56-58, 66 and 69 (zur Hausen, 1996). It should be clear to one skilled in the art that other probes than those listed in table 7 or 12 may be chosen within said region D, provided that they specifically hybridize to only one HPV-type. It should also be clear that in some cases probes may be chosen that overlap with the primers used in the amplification step. In this case, however, the region of overlap between primer and probe should not be as long as to allow by itself duplex formation under the experimental conditions used. It should furthermore be clear that, if presently unknown types are detected that differ in the D region from all presently known types, the methods of this invention will also enable detection and/or identification of said presently unknown HPV types. The present invention furthermore discloses novel sequences in said 22 bp region, as shown in example 5 and in FIG. 1 (SEQ ID NO 135-153). Probes or primers that are designed to specifically hybridize to these sequences, may be used in a method to detect and/or to identify HPV polynucleic acids comprising any of these sequences, when these polynucleic acids are present in a biological sample.
According to another preferred embodiment of the present invention, probes are used that specifically hybridize to the D region, or more particularly to the E region of more than one HPV type. Examples of such probes are given in table 9 and in table 10. The probes in table 9 have been designed for hybridization in a microtiter plate, e.g. according to the DEIA technique (see below), whereas the probes in table 10 are more suitable for the LiPA technique (see below). These probes hybridize to the E region of more than one HPV type, and hence may be used to detect the presence in a biological sample of any of the types to which they hybridize. It should be clear to one skilled in the art that, according to this embodiment, other probes than those listed in table 9 and table 10 may be chosen within the D region, provided that they hybridize to one or more than one HPV type.
According to another preferred embodiment of the present invention, the aforementioned methods of detection and/or identification of HPV are characterized further in that the hybridization step involves a reverse hybridization format. This format implies that the probes are immobilized to certain locations on a solid support and that the amplified HPV polynucleic acids are labelled in order to enable the detection of the hybrids formed. According to this embodiment, at least one probe, or a set of a least 2, preferably at least 3, more preferably at least 4 and most preferably at least 5 probes is used. When at least 2 probes are used, said probes are meticulously designed in such a way that they specifically hybridize to their target sequences under the same hybridization and wash conditions.
According to an even more preferred embodiment of the present invention, the aforementioned hybridization step is performed according to the LiPA technique. Said technique involves a reverse hybridization assay, characterized in that the oligonucleotide probes are immobilized on a solid support as parallel lines (Stuyver et al., 1993; international application WO 94/12670). The reverse hybridization format and particularly the LiPA format have many practical advantages as compared to other DNA techniques or hybridization formats, especially when the use of a combination of probes is preferable or unavoidable to obtain the relevant information sought.
Alternatively, detection of HPV polynucleic acids in a biological sample may be performed by use of the DNA Enzyme Immuno Assay (DEIA). This method is used for rapid and specific detection of PCR products. PCR products are generated by a primer set, of which either the forward or the reverse primer contain biotin at the 5xe2x80x2 end. This allows binding of the biotinylated amplimers to streptavidin-coated microtiter wells. PCR products are denatured by sodium hydroxide, which allows removal of the non-biotinylated strand. Specific labelled oligonucleotide probes (e.g. with digoxigenin) are hybridized to the single-stranded immobilized PCR product and hybrids are detected by enzyme-labelled conjugate and calorimetric methods.
The present invention also relates to sets of oligonucleotides, said sets comprising at least one primer and/or at least one probe that may be used to perform the methods for detection and/or identification of HPV as described above. Preferred primers according to the present invention can for instance be chosen from table 1, table 4 and table 11. Preferred probes are shown in tables 7, 9, 10 and 12. These probes can be optimized to be used together in a given format, e.g. a LiPA format, under the same hybridization and washing conditions. Evidently, when other hybridization conditions would be preferred, all probes should be adapted accordingly by adding or deleting one or more nucleotides at their extremities. It should be understood that these concomitant adaptations should give rise to the same result, namely that the probes still hybridize specifically to their respective type-specific target sequences. Such adaptations may also be necessary if the amplified material is RNA and not DNA as is the case in the NASBA system.
The present invention also relates to diagnostic kits for detection and/or identification of HPV, possibly present in a biological sample, comprising the following components:
(i) at least one suitable primer or at least one suitable primer pair;
(ii) at least one suitable probe, preferably at least 2, more preferably at least 3, even more preferably at least 4 and most preferably at least 5 suitable probes, possibly fixed to a solid support;
(iii) a hybridization buffer, or components necessary for the production of said buffer, or instructions to prepare said buffer;
(iv) a wash solution, or components necessary for the production of said solution, or instructions to prepare said solution;
(v) optionally a means for detection of the hybrids formed;
(vi) optionally a means for attaching the probe(s) to a known location on a solid support.
The following definitions and explanations will permit a better understanding of the present invention.
HPV isolates that display a sequence difference of more than 10% to any previously known type in the combined nucleotide sequences of E6, E7 and L1 genes (Chan et al., 1995, de Villiers, 1994) are classified as different HPV xe2x80x9cgenotypesxe2x80x9d. HPV isolates that differ between 2 and 10% are classified as different xe2x80x9csubtypesxe2x80x9d. If the sequence variation is below 2%, the isolates are classified within the same subtype as different xe2x80x9cvariantsxe2x80x9d. The term xe2x80x9ctypexe2x80x9d when applied to HPV refers to any of the three categories defined above.
The target material in the samples to be analyzed may either be DNA or RNA, e.g. genomic DNA, messenger RNA, viral RNA or amplified versions thereof. These molecules are in this application also termed xe2x80x9cpolynucleic acidsxe2x80x9d.
Well-known extraction and purification procedures are available for the isolation of RNA or DNA from a sample (e.g. in Sambrook et al.,1989).
The term xe2x80x9cprobexe2x80x9d according to the present invention refers to a single-stranded oligonucleotide which is designed to specifically hybridize to HPV polynucleic acids.
The term xe2x80x9cprimerxe2x80x9d refers to a single stranded oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions at which the primer is used, such as temperature and ionic strength.
The expression xe2x80x9csuitable primer pairxe2x80x9d in this invention refers to a pair of primers allowing the amplification of part or all of the HPV polynucleic acid fragment for which probes are immobilized.
The term xe2x80x9ctarget sequencexe2x80x9d of a probe or a primer according to the present invention is a sequence within the HPV polynucleic acids to which the probe or the primer is completely complementary or partially complementary (i.e. with some degree of mismatch). It is to be understood that the complement of said target sequence is also a suitable target sequence in some cases. Probes of the present invention should be complementary to at least the central part of their target sequence. In most cases the probes are completely complementary to their target sequence. The term xe2x80x9ctype-specific target sequencexe2x80x9d refers to a target sequence within the polynucleic acids of a given HPV type that contains at least one nucleotide difference as compared to any other HPV-type.
xe2x80x9cSpecific hybridizationxe2x80x9d of a probe to a region of the HPV polynucleic acids means that, after the amplification step, said probe forms a duplex with part of this region or with the entire region under the experimental conditions used, and that under those conditions said probe does not form a duplex with other regions of the polynucleic acids present in the sample to be analysed. It should be understood that probes that are designed for specific hybridization to a region of HPV polynucleic acids, may fall within said region or may to a large extent overlap with said region (i.e. form a duplex with nucleotides outside as well as within said region). For instance, some of the probes that are shown in table 7 and that are designed for specific hybridization to the 22 bp region between the B and the C regions (FIG. 1), extend up to 5 nucleotides beyond the 3xe2x80x2-end of said 22 bp region and other probes of table 7 extend up to 3 nucleotides beyond the 5xe2x80x2-end of said 22 bp region.
xe2x80x9cSpecific hybridizationxe2x80x9d of a primer to a region of the HPV polynucleic acids means that, during the amplification step, said primer forms a duplex with part of this region or with the entire region under the experimental conditions used, and that under those conditions said primer does not form a duplex with other regions of the polynucleic acids present in the sample to be analysed. It should be understood that primers that are designed for specific hybridization to a region of HPV polynucleic acids, may fall within said region or may to a large extent overlap with said region (i.e. form a duplex with nucleotides outside as well as within said region).
Since the current application requires the detection of single base pair mismatches, stringent conditions for hybridization of probes are required, allowing only hybridization of exactly complementary sequences. However, it should be noted that, since the central part of the probe is essential for its hybridization characteristics, possible deviations of the probe sequence versus the target sequence may be allowable towards the extremities of the probe when longer probe sequences are used. Variations are possible in the length of the probes. Said deviations and variations, which may be conceived from the common knowledge in the art, should however always be evaluated experimentally, in order to check if they result in equivalent hybridization characteristics as the exactly complementary probes.
Preferably, the probes of the invention are about 5 to 50 nucleotides long, more preferably from about 10 to 25 nucleotides. Particularly preferred lengths of probes include 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics.
Probe sequences are represented throughout the specification as single stranded DNA oligonucleotides from the 5xe2x80x2 to the 3xe2x80x2 end. It is obvious to the man skilled in the art that any of the below-specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).
The probes according to the invention can be prepared by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by excision of the latter from the cloned plasmids by use of the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, for instance by the conventional phospho-triester method.
The fact that amplification primers do not have to match exactly with the corresponding target sequence in the template to warrant proper amplification is amply documented in the literature (Kwok et al., 1990). However, when the primers are not completely complementary to their target sequence, it should be taken into account that the amplified fragments will have the sequence of the primers and not of the target sequence. Primers may be labelled with a label of choice (e.g. biotine). The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu and Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of Qxcex2 replicase (Lizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules known in the art.
The oligonucleotides used as primers or probes may also comprise nucleotide analogues such as phosphorothiates (Matsukura et al., 1987), alkylphosphorothiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984). As most other variations or modifications introduced into the original DNA sequences of the invention these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity. However the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides. The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.
The term xe2x80x9csolid supportxe2x80x9d can refer to any substrate to which an oligonucleotide probe can be coupled, provided that it retains its hybridization characteristics and provided that the background level of hybridization remains low. Usually the solid substrate will be a microtiter plate (e.g. in the DEIA technique), a membrane (e.g. nylon or nitrocellulose) or a microsphere (bead) or a chip. Prior to application to the membrane or fixation it may be convenient to modify the nucleic acid probe in order to facilitate fixation or improve the hybridization efficiency. Such modifications may encompass homopolymer tailing, coupling with different reactive groups such as aliphatic groups, NH2 groups, SH groups, carboxylic groups, or coupling with biotin, haptens or proteins.
The term xe2x80x9clabelledxe2x80x9d refers to the use of labelled nucleic acids. Labelling may be carried out by the use of labelled nucleotides incorporated during the polymerase step of the amplification such as illustrated by Saiki et al. (1988) or Bej et al. (1990) or labelled primers, or by any other method known to the person skilled in the art. The nature of the label may be isotopic (32P, 35S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
The xe2x80x9csamplexe2x80x9d may be any biological material taken either directly from the infected human being (or animal), or after culturing (enrichment). Biological material may be e.g. is scrapes or biopsies from the urogenital tract or any part of the human or animal body.
The sets of probes of the present invention will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13,14,15,16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more probes. Said probes may be applied in two or more (possibly as many as there are probes) distinct and known positions on a solid substrate. Often it is preferable to apply two or more probes together in one and the same position of said solid support.
For designing probes with desired characteristics, the following useful guidelines known to the person skilled in the art can be applied.
Because the extent and specificity of hybridization reactions such as those described herein are affected by a number of factors, manipulation of one or more of those factors will determine the exact sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The importance and effect of various assay conditions are explained fisher herein.
**The stability of the [probe:target] nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long AT-rich sequences, by terminating the hybrids with G:C base pairs, and by designing the probe with an appropriate Tm. The beginning and end points of the probe should be chosen so that the length and % GC result in a Tm about 2-10xc2x0 C. higher than the temperature at which the final assay will be performed. The base composition of the probe is significant because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs due to additional hydrogen bonding. Thus, hybridization involving complementary nucleic acids of higher G-C content will be more stable at higher temperatures.
**Conditions such as ionic strength and incubation temperature under which a probe will be used should also be taken into account when designing a probe. It is known that the degree of hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of the hybrids will increase with increasing ionic strength. On the other hand, chemical reagents, such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, will increase the stringency of hybridization. Destabilization of the hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5xc2x0 C. below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity.
**It is desirable to have probes which hybridize only under conditions of high stringency. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. The degree of stringency is chosen such as to maximize the difference in stability between the hybrid formed with the target and the nontarget nucleic acid. In the present case, single base pair changes need to be detected, which requires conditions of very high stringency.
**The length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly complementary base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, preferred oligonucleotide probes of this invention are between about 5 to 50 (more particularly 10-25) bases in length and have a sufficient stretch in the sequence which is perfectly complementary to the target nucleic acid sequence.
**Regions in the target DNA or RNA which are known to form strong internal structures inhibitory to hybridization are less preferred. Likewise, probes with extensive self-complementarity should be avoided. As explained above, hybridization is the association of two single strands of complementary nucleic acids to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. There can be intramolecular and intermolecular hybrids formed within the molecules of one type of probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. Computer programs are available to search for this type of interaction. However, in certain instances, it may not be possible to avoid this type of interaction.
**Standard hybridization and wash conditions are disclosed in the Materials and Methods section of the Examples. Other conditions are for instance 3xc3x97SSC (Sodium Saline Citrate), 20% deionized FA (Formamide) at 50xc2x0 C. Other solutions (SSPE (Sodium saline phosphate EDTA), TMAC (Tetramethyl ammonium Chloride), etc.) and temperatures can also be used provided that the specificity and sensitivity of the probes is maintained. When needed, slight modifications of the probes in length or in sequence have to be carried out to maintain the specificity and sensitivity required under the given circumstances.
In order to identify different HPV types with the selected set of oligonucleotide probes, any hybridization method known in the art can be used (conventional dot-blot, Southern blot, sandwich, etc.). However, in order to obtain fast and easy results if a multitude of probes are involved, a reverse hybridization format may be most convenient. In a preferred embodiment the selected probes are immobilized to a solid support in known distinct locations (dots, lines or other figures). In another preferred embodiment the selected set of probes are immobilized to a membrane strip in a line fashion. Said probes may be immobilized individually or as mixtures to delineated locations on the solid support. A specific and very user-friendly embodiment of the above-mentioned preferential method is the LiPA above-mentioned set of probes is immobilized in parallel lines on a membrane, as further described in Example 4. The HPV polynucleic acids can be labelled with biotine, and the hybrid can then, via a biotine-streptavidine coupling. be detected with a non-radioactive colour developing system.
The term xe2x80x9chybridization bufferxe2x80x9d means a buffer allowing a hybridization reaction between the probes and the polynucleic acids present in the sample, or the amplified products, under the appropriate stringency conditions.
The term xe2x80x9cwash solutionxe2x80x9d means a solution enabling washing of the hybrids formed under the appropriate stringency conditions.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and variations such as xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of stated integers or steps but not to the exclusion of any other integer or step or group of integers or steps.